The invention relates to a method and apparatus for guiding and collecting light which issues from a light source and which is passed as an incident primary beam to a specimen or like material to be measured, by which a measuring beam leaving the material to be measured is conducted to at least one detector or the like photoelectric receiver.
When measuring radiation outputs, the signal amplitude of the detector is directly dependent on the intensity of the light to be measured. In that procedure, however, signal noise occurs, produced by the light and the detector, which, because of the random nature of the noise, rises in a sub-proportional fashion relative to the total light detected (the noise increases approximately with .sqroot.1/J). Therefore, because of severe noise, very weak signals or very slight changes in a signal cannot be detected, or can only be detected with difficulty, using expensive technical equipment and long measuring integration times. It is therefore crucial for the light output of the system to be maximized in order to produce a good signal-to-noise ratio.
Very weak signals are produced primarily in the area of luminescence measuring operations. Luminescence phenomena are based on the conversion of absorbed energy into light radiation by suitable molecules. When energy is absorbed, the molecule to be observed jumps to a higher level of oscillation. The excitation energy used is generally a high-energy primary light beam, the wavelength of which differs from that of the measuring beam. Within a period of about 10-12 seconds, the excited molecule gives off a large part of the absorbed energy again by impact with the adjacent molecule. A small part of the absorbed energy can be given off again by the molecule in the form of photons in any direction, and can then be quantified by a measuring means which is capable of selectively detecting the light which is produced in that way. Generally, the degrees of intensity of the primary beam and the measuring beam differ by powers of ten.
Conventional light measuring means detect the measuring light which is irradiated in all directions only within a solid angle of a few degrees (2 to 3 degrees). The remainder of the light remains unused, and can in fact interfere with the measuring operation, in the form of stray or spurious light. Although the measuring arrangement may be of such a configuration, at high technical expense, that so-called single photon counting is made possible, such equipment is however unsuitable for routine measuring operations as it requires long measuring times and is technically exacting.
In luminometry, an increase in the intensity of the primary light does not result in a clear improvement in the measuring operation as only a small fraction of the input energy can be given off again in the form of luminescence. In contrast, high levels of intensity of primary light increase the problems of selectively detecting the secondary or measuring light as the stray or spurious light components are increased at the same time. Likewise, an unsuitable way of improving the measuring operation is to increase the concentration of luminescing molecules, as with an increasing level of concentration, the so-called concentration extinction phenomenon (saturation) becomes more and more noticeable. The more highly concentrated a solution of excited molecules is, the more frequently does an excited molecule lose all its energy by impact against adjacent molecules, before it can emit light. Therefore, it is only in respect of heavily diluted solutions and constant primary light that the level of intensity of a given fluorescence wavelength is proportional to the concentration of the fluorescing substance.
Luminescence measuring operations are primarily used nowadays in the area of clinical research and analysis. However, the methods of investigation are subject to limits by virtue of the inadequate degree of sensitivity of routine measuring equipment, as is required for measuring levels of physiological concentration in the biologically clinical area. Great efforts are being made at this time to replace radioimmunoassays (RIA) which rePresent a health risk from the point of view of the laboratory personnel, by fluorescence immunoassays (FIA). FIAs represent a method which is comparable to RIAs, in regard to its theoretical sensitivity but which has never achieved the expected breakthrough in the laboratory in the absence of suitable routine equipment. The present invention seeks to contribute to improving that situation.
Very weak and in particular imprecise signals also occur in absorption measuring operations in respect of media which have a strong light-scatter of diffusion effect. As conventional photometers only detect the light which passes in a straight line through the specimen or sample to be measured, and the attenuation of such light, it is often impossible to measure the actual light absorption of such media. Stray or scatter light problems occur in absorption measuring operations in respect of colloid-like substances such as photographic emulsions, solutions with macromolecules or polymers, or oily liquids. The present invention seeks to contribute to improving that situation.
When a light source such as an arc lamp is used for illumination purposes, it is usually interposed between a spherical mirror and a lens, the two being arranged in such a way that the arc lamp is in their focal point. This leads to quasi-parallel light which strictly speaking is only parallel for one particular wavelength of light and is disturbed by the spherical aberration of the lens. In addition, only a relatively small solid angle of the light can be used. It is therefore difficult to reach high density light of different wavelengths (or composed light) by focusing the light arising out of such a system afterwards by lenses. An alternative technique which is used is the application of lasers as light sources of high luminous density. This technique suffers the disadvantages of low stability, limited wavelength range and high costs. While it is relatively easy to stabilize the total output of an arc lamp to 1/1000 in relative intensity units, it is not possible to produce strictly parallel light as mentioned above. There is a lack of light source combining high stability, large wavelength range and highly parallel radiation output which is highly desirable for certain applications like microfluorimetry and fluorimetry in general and photo-bleaching or photo-activation in the microscopic range. The present invention allows one to construe a light source fulfilling the above desirable characteristics.